重组慢病毒介导RNA干扰抑制DNA依赖蛋白激酶催化亚基表达对肺癌细胞中C225诱导表皮生长因子受体核转位及敏感性的影响  

The effect of lentivirus-mediated small interfering RNA interference targeting DNA-PKcs on epidermal growth factor receptor translocation and sensitivity to C225 in lung cancer cell line

作　　者：潘燚[1] 姚陈[2] 刘大钺[2] 李伟雄[1] 张红丹[1] 杨素清[3] 

机构地区：[1]广东省医学科学院广东省人民医院肿瘤中心放疗科,广州510080 [2]中山大学附属第一医院普通外科 [3]广东省医学科学院广东省人民医院医学研究中心,广州510080

出　　处：《中华实验外科杂志》2014年第8期1626-1628,共3页Chinese Journal of Experimental Surgery

基　　金：广东省自然科学基金资助项目（S2011010000791）;广东省医学科研基金资助项目（A2011026）

摘　　要：目的 构建人DNA依赖蛋白激酶催化亚基（DNA-PKcs）基因RNA干扰慢病毒载体,观察在非小细胞肺癌细胞株中沉默DNA-PKcs对C225诱导的表皮生长因子受体（EGFR）核转位及C225抑制细胞增殖的影响.方法 构建慢病毒干扰载体（LV-si-DNA-PKcs）感染A549细胞.荧光定量聚合酶链反应（FQ-PCR）检测DNA-PKcs mRNA表达;Western blot检测DNA-PKcs和细胞核EGFR蛋白的表达.细胞计数试剂盒（CCK-8）法检测C225对细胞的增殖抑制作用.结果 LV-si-DNA-PKcs感染的A549细胞见绿色荧光,感染率达95％以上;LV-si-DNA-PKcs有效干扰DNA-PKcs蛋白及mRNA表达水平（P＜0.05）.加C225前未见细胞核EGFR表达.C225处理1h,LV-si-DNA-PKcs细胞株见细胞核EGFR表达,持续用药4、24 h细胞核EGFR表达无明显增加;在LV-si-control细胞株及空白组,C225处理1、4、24h后细胞核EGFR表达逐渐增多.C225处理48 h后,LV-si-DNA-PKcs细胞株的细胞存活率开始低于其他2组细胞,72 h细胞存活率明显降低[（40.04±2.89）％比（55.82±7.11）％、（52.67±1.43）％,F=9.392,P＜0.01].结论 抑制A549细胞中DNA-PKcs表达,在一定程度上抑制了C225诱导的EGFR细胞核转位,增强C225对细胞的增殖抑制作用.Objective Intrinsic and acquired resistances to cetuximab （C225）,the anti-epidermal growth factor receptor （EGFR） monoclonal antibody,limit its clinical application.After nuclear translocation,EGFR combines with DNA-dependent protein kinase catalytic subunit （DNA-PKcs） and then functions as a co-transcriptional and regulatory factor to enhance resistance to C225.This study was to discuss the effect of Lentivirus-mediated small interfering RNA （siRNA） interference targeting DNA-PKcs on EGFR translocation induced by C225 and cellular proliferation inhibition of C225 in lung cancer cell line.Methods The lentiviral vector expressing DNA-PKcs small interfering RNA （LV-si-DNA-PKcs） was constructed to suppress DNA-PKcs expression in A549 cell line.The targeted gene expression was detected by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）.The contents of DNA-PKcs and EGFR were measured by Western blotting.Cell counting kit-8 （CCK-8） was used to determine the inhibitory effect of C225 on cell proliferation at different time points.Results A549 cells infected with LV-siDNA-PKcs were GFP-positive and the delivery efficiency was more than 95％.After lentivirus mediated siRNA transfection,the DNA-PKcs expressions on mRNA and protein levels in LV-si-DNA-PKcs infected cells were significantly reduced compared with control （LV-si-control） and blank groups （P ＜ 0.05）.Before C225 treatment,there were no nuclear EGFR expressions in the 3 group cells.After 1 hour C225 treatment,nuclear EGFR expression increased in LV-si-DNA-PKcs infected cells,but it kept stable after 4 and 24 hours.In control and blank groups,nuclear EGFR expressions continued rising after C225 treatment.With C225 treatment for 48 hours,cell survival rate in LV-si-DNA-PKcs infected cells began falling and significantly decreased in 72 hours compared to other groups [（40.04 ± 2.89） ％ vs.（55.82 ± 7.11） ％ and （52.67 ±1.43）％,F=9.392,P＜0.01].Conclusion Our findings suggest that suppress

关 键 词：DNA依赖蛋白激酶催化亚基 细胞核表皮生长因子受体 西妥昔单抗 

分 类 号：R734.2[医药卫生—肿瘤]